LISTERIA HEMOLYSIN AND ESCAPE FROM A VACUOLE

李斯特菌溶血素和从液泡中逃逸

• 批准号: 2699928
• 负责人: DANIEL A PORTNOY
• 金额: $ 24.54万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-15 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2699928
• 关键词: Escherichia coli; Listeria; Listeria infections; acid base balance; antigen presentation; hemolysin; host organism interaction; intracellular parasitism; macrophage; molecular cloning; phagocytosis; plasmids; pore forming protein; posttranslational modifications; protein sequence; recombinant proteins; site directed mutagenesis; tissue /cell culture; transfection /expression vector; vesicle /vacuole; virulence

DESCRIPTION (Adapted from the applicant's abstract): Listeria monocytogenes is a model facultative intracellular pathogen which primarily infects pregnant women and immunocompromised individuals. A primary determinant of L. monocytogenes pathogenesis is a secreted pore-forming protein referred to as listeriolysin O (LLO). LLO is largely responsible for rupture of the host vacuole which results from phagocytosis. Perfringolysin O (PFO) is a related pore-forming protein which is involved in the pathogenesis of infections by an extracellular pathogen. When expressed by L. monocytogenes, PFO mediates escape from the phagocytic vacuole, but is toxic to the cell. Normally, LLO is continually expressed during infection, but in contrast to PFO, it is proteolytically degraded in the cytosol and presented on the cell surface in association with MHC class I molecules. It is hypothesized that pH optimum and cytosolic processing are essential LLO-encoded determinants which distinguish it from PFO. The focus of the current proposal is to define the precise structural and mechanistic features of LLO which differentiate it from other members of the family of thiol-activated cytolysins, facilitate its intravacuolar activity, and direct its fate in the cytosol. In Aim I, protein sequences responsible for LLO's acidic pH optimum and processing in the host cytosol will be identified. This will be accomplished by domain and sub-domain swapping between LLO and PFO, and modified charged-to-alanine scanning mutagenesis. The LLO/PFO chimeras will be purified from E. coli and characterized biochemically. Next, the chimeras will be introduced into L. monocytogenes and characterized in tissue culture models of infection., In Aim II, the pathway of LLO processing in the host cytosol will be fully evaluated. LLO will be identified by metabolic labeling of L. monocytogenes within infected host cells, followed by immunoprecipitation. Precursor/product relationships will be determined by pulse-chase experiments. Specific inhibitors will be used to evaluate the role of the proteosome and other proteases in degradation. The role of LLO phosphorylation will be evaluated biochemically and genetically. In Aim III, the precise nature of the L. monocytogenes phagosome will be characterized with regard to pH, time of perforation, and markers of the endosome/lysosome pathway of maturation. The role of pH optimum and the L. monocytogenes phospholipases will be evaluated by using mutant and chimeric strains. In Aim IV, the investigators will evaluate the feasibility of using E. coli K12 expressing LLO and a recombinant protein as a novel system to introduce foreign proteins into the mammalian cytosol for antigen presentation.

描述（改编自申请人的摘要）：单核细胞增生李斯特氏菌 是一种模型兼性细胞内病原体，主要感染 孕妇和免疫功能低下的人。 的一个主要决定因素 单核细胞增生李斯特氏菌的发病机制是一种分泌性成孔蛋白，称为 如李斯特菌溶血素 O (LLO)。 LLO 是导致破裂的主要原因 由吞噬作用产生的宿主液泡。 产气荚膜溶血素 O (PFO) 是一种 相关成孔蛋白，参与发病机制 细胞外病原体感染。 当用L表示时。 单核细胞增多症，PFO 介导吞噬泡的逃逸，但具有毒性 到细胞。 通常情况下，LLO 在感染期间持续表达，但是 与 PFO 相比，它在细胞质中被蛋白水解降解，并且 与 MHC I 类分子相关地呈现在细胞表面。 它 假设最佳 pH 值和胞质处理至关重要 LLO 编码的决定因素将其与 PFO 区分开来。 当前提案的重点是定义精确的结构和 LLO 区别于其他成员的机制特征 硫醇激活的溶细胞素家族，促进其液泡内活性， 并在细胞质中指导其命运。 在目标 I 中，负责的蛋白质序列 对于 LLO 的酸性 pH 最佳值和在宿主细胞质中的加工将是 确定。 这将通过域和子域交换来完成 LLO 和 PFO 之间的差异，以及改进的带电至丙氨酸扫描诱变。 LLO/PFO 嵌合体将从大肠杆菌中纯化并进行表征 生物化学上。 接下来，嵌合体将被引入单核细胞增生李斯特菌中 并在感染的组织培养模型中进行表征。 在目标 II 中，宿主细胞质中 LLO 加工的途径将完全被 评价。 LLO 将通过单核细胞增生利斯特氏菌的代谢标记来识别 在受感染的宿主细胞内，然后进行免疫沉淀。 前体/产物关系将由脉冲追踪确定 实验。 将使用特定抑制剂来评估其作用 蛋白酶体和其他蛋白酶的降解。 LLO 的作用 将对磷酸化进行生化和遗传学评估。 在目标 III 中，单核细胞增生李斯特氏菌吞噬体的确切性质将是 特征包括 pH 值、穿孔时间和标记物 内体/溶酶体成熟途径。 最适 pH 值和 L 的作用。 单核细胞增多性磷脂酶将通过使用突变体和嵌合体进行评估 菌株。 在目标 IV 中，研究人员将评估使用大肠杆菌的可行性 K12 表达 LLO 和重组蛋白作为新系统来引入 外源蛋白进入哺乳动物细胞质以进行抗原呈递。